Apoptosis, or programmed cell death (PCD), is a genetically regulated mechanism with a central role in both metazoan development and homeostasis. (Raff, 1992; Steller, 1995). The cell death machinery is conserved throughout evolution (Vaux et al., 1994) and is composed of several distinct parts including effectors, inhibitors and activators (Chinnaiyan and Dixit, 1996; Steller, 1995). Invertebrate model systems have been invaluable in identifying and characterizing the genes that control apoptosis. (Hengartner, 1996). While numerous candidate genes have been identified, how they interact to execute the apoptotic program is poorly understood.
It is becoming apparent that cysteine proteases related to the Caenorhabditis elegans cell death gene ced-3 represent the effector components of the apoptotic machinery. The first mammalian homolog of CED-3 identified was interleukin-1.beta. converting enzyme (ICE). (Yuan et al., 1993). Overexpression of ICE or CED-3 in Rat-1 fibroblasts induced apoptosis, suggesting that ICE was functionally, as well as structurally, related to CED-3. (Miura et al., 1993). However, such evidence is only a correlation, as ectopic expression of a number of proteases, including chymotrypsin, proteinase K and trypsin, cause significant apoptosis. (Williams and Henkart, 1994).
Further studies suggested that proteases related to ICE, rather than ICE itself, may play a more important role in the apoptotic mechanism. First, a number of cell types stably secrete mature IL-1.beta. without undergoing apoptosis. Second, ICE-deficient mice, although unable to generate active IL-1.beta., fail to exhibit a prominent cell death defective phenotype,(Kuida et al., 1995; Li et al., 1995). Third, in an in vitro model of apoptosis, condemned phase extracts prepared from chicken DU249 cells failed to cleave the primary substrate of ICE, pro-IL-1b. (Lazebnik et al., 1994). Instead, a proteolytic activity in these extracts, termed prICE, cleaved the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) into signature apoptotic fragments. (Lazebnik et al., 1994). Purified ICE failed to cleave PARP (Lazebnik et al., 1994; Tewari et al., 1995), suggesting that prICE was distinct from ICE.
To date, seven homologs of CED-3 and ICE have been characterized and include Nedd-2/ICH1. (Kumar et al., 1994; Wang et al., 1994), Yama/CPP32/apopain. (Fernandes-Alnemri et al., 1994; Nicholson et al., 1995; Tewari et al., 1995), Tx/ICH2/ICE rel-II. (Faucheu et al., 1995; Kamens et al., 1995; Munday et al., 1995), ICE rel-III. (Munday et al., 1995), Mch2. (Fernandes-Alnemri et al., 1995a), ICE-LAP3/Mch3/CMH-1 . (Duan et al., 1996a; Fernandes-Alnemri et al., 1995b; Lippke et al., 1996) and ICE-LAP6. (Duan et al., 1996b). Ectopic expression of these ICE/CED-3 homologs in a variety of cells causes apoptosis. Only Yama and ICE-LAP3 have been shown to be proteolytically activated by apoptotic stimuli (Chinnaiyan et al., 1996a; Duan et al., 1996a; Schlegel et al., 1996). Future studies will delineate which family members have an important role in the apoptotic mechanism.
Although it is clear that CED-3-like proteases are distal effectors of the cell death pathway, the proximal components that mediate their activation remain to be identified. Two cell surface cytokine receptors, CD95 (Fas/APO-1) and TNFR-1, have been shown to trigger apoptosis by their natural ligands or specific agonist antibodies. (Baglioni, 1992; Itoh et al., 1991; Trauth et al., 1989). Both death receptors are members of the tumor necrosis factor (TNF)/ nerve growth factor (NGF) receptor family which also include TNFR-2, low-affinity NGFR, CD40 and CD30 among others,(Smith et al., 1990; Tewari and Dixit, 1995). While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, CD95 and TNFR-1 also share a region of homology, appropriately designated the "death domain", required to signal apoptosis. (Itoh and Nagata, 1993; Tartaglia et al., 1993). This shared "death domain" suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified. Using the two-hybrid system, three death domain-containing molecules, TRADD, FADD/MORT1 and RIP, were isolated. (Boldin et al., 1995; Chinnaiyan et al., 1995; Cleveland and Ihle, 1995; Hsu et al., 1995; Stanger et al., 1995). Subsequent studies showed that endogenous FADD associates with CD95 in an activation-dependent fashion. (Kischkel et al., 1995), while similarly, endogenous TRADD and RIP were found complexed to activated TNFR-1 (Hsu et al., 1996a; Hsu et al., 1996b). It has been postulated that TRADD acts as an adaptor molecule for TNFR-1. (Hsu et al., 1996a), mediating the interaction of TNFR-1 with FADD, while, by contrast, RIP may be involved in NF-kB signaling. (Hsu et al., 1996b). A dominant negative version of FADD (FADD-DN) blocks TNF- and CD95-induced apoptosis, suggesting that FADD functions as the common signaling conduit for cytokine-mediated cell death. (Chinnaiyan et al., 1996a; Hsu et al., 1996a).
The first evidence for the involvement of ICE-like proteases in CD95- and TNFR-1 signaling came with the discovery that the poxvirus gene product CrmA blocks cell death triggered by both receptors,(Enari et al., 1995; Los et al., 1995; Tewari and Dixit, 1995b). In vitro, the serpin CrmA interacts only with the active forms of ICE and ICE-like proteases (Ray et al., 1992; Tewari et al., 1995). Yama and ICE-LAP3, two of the ICE-like enzymes most related to CED-3, are expressed as zymogens that are proteolytically activated upon ligation of CD95 or TNFR-1 (Chinnaiyan et al., 1996b; Duan et al., 1996a). However, both Yama and ICE-LAP3 remained as proenzymes in anti-CD95 treated CrmA-expressing cells, suggesting that CrmA inhibits an ICE-like protease upstream of Yama and ICE-LAP3 (Chinnaiyan et al., 1996b). The mechanism by which the death receptors engage the cytosolic apoptotic proteases is of central importance to cell death research.
Activation of CD95 initiates association with at least four proteins designated CAP, for cytotoxicity-dependent APO-1-associated proteins. (Kischkel et al., 1995). CAP1 and CAP2 were identified as alternate forms of serine phosphorylated FADD, while the identity of the other CAPs, CAP3 and CAP4, remains to be determined. The four associating proteins, along with the oligomerized receptor, have been termed the CD95 death-inducing signaling complex (DISC). (Kischkel et al., 1995). Later studies demonstrated that a dominant negative version of FADD, missing the N-terminal death effector domain (DED), functions by blocking the recruitment of CAP3 and CAP4 to the DISC. (Chinnaiyan et al., 1996a). Thus, these results strongly suggest that CAP3 and CAP4 are downstream components of the CD95 signaling cascade.
Recently, the method of protein sequencing using electrospray (Fenn et al., 1989), in combination with tandem mass spectrometry. (Hunt et al., 1986), was refined to allow sequencing of femtomole quantities of proteins directly isolated from silver stained gels (Wilm et al., 1996). The peptide sequence tags (Mann and Wilm, 1994) generated can then be used to screen sequence databases to obtain matching sequences leading to isolation of full-length clones for functional characterization. In its first reported use, an anti-angiogenic factor derived from mycoplasma was characterized using this technique. (Wilm et al., 1996).
To identify CAP3 and CAP4, we took advantage of the above technology of nano-electrospray tandem mass spectrometry (nanoES MS/MS). CAP4 is a novel cysteine protease of the ICE/CED-3 family which contains a pro-domain homologous to FADD and has therefore been designated FLICE (for FADD-Like ICE) or ICE LAP-7. CAP3 was identified as the pro-domain of FLICE likely generated during CD95-induced proteolytic activation. In vitro, the DED of FADD can directly bind FLICE. Thus, these results suggest that CD95 utilizes the adaptor protein FADD to physically engage FLICE, the apical component of a proteolytic cascade made up of other ICE/CED-3 -like proteases.
Results
Identification of FLICE (CAP4/CAP3)
At least four endogenous proteins, CAP 1-4 (for cytoxicity-dependent APO-1-associated proteins), associate with activated CD95 to form the DISC. (Kischkel et al., 1995). CAP1 and CAP2 were identified as different forms of the previously isolated FADD/MORT1. (Kischkel et al., 1995). Subsequent dominant negative studies established that endogenous FADD is essential for the recruitment of CAP3 and CAP4 to the CD95 DISC (Chinnaiyan et al., 1996a). To identify CAP4, we utilized nano-electrospray mass spectrometry. (Mann and Wilm, 1995; Wilm and Mann, 1996) to generate peptide sequence from gel-isolated protein. The CAP4 spot contained approximately 0.5 pmol of protein. The complete sequence of 5 peptides covering a total of 41 amino acid residues and two partially sequenced peptides which could be used as sequence tags were determined (FIG. 1A). Homology searches against a comprehensive non-redundant database (NRDB, maintained by C. Sander, EMBL) revealed no matches to known proteins. However, when the Human Genome Sciences cDNA database was searched, a 3.0 kb cDNA was identified and found to contain a 1437-base pair open reading frame that encoded a novel protein with a predicted molecular mass of 55.3 kDa and a pI of 4.91, consistent with the size and the pI of CAP4 as determined by 2-D gel analysis. The putative initiator methionine (AAGATGG) was in agreement with the consensus Kozak's sequence for translation initiation. A BLAST search of the GenBank protein database revealed that the novel cDNA, designated FLICE, had substantial homology to both FADD and the ICE/CED-3 family of cysteine proteases. CAP3 was also subjected to nanoES MS/MS. Two peptides were sequenced, both being identical to two peptides (T2 and T4) found in the sequencing of the total tryptic digest of CAP4 (FIG. 1A). Therefore, CAP3 represents an N-terminal fragment of FLICE.
FLICE has Homology to the DED of FADD
The association of CD95 with FADD is mediated by their respective C-terminal death domains. (Boldin et al., 1995; Chinnaiyan et al., 1995). A truncated derivative of FADD (FADD-DN), which contains the death domain but lacks the N-terminus, functions as a potent dominant negative regulator of CD95-induced apoptosis. (Chinnaiyan et al., 1996a). By contrast, the 117 N-terminal amino acids of FADD are capable of triggering apoptosis and, therefore, this segment has been termed the death effector domain (DED) (Chinnaiyan et al., 1995). Interestingly, FLICE contains two N-terminal stretches of approximately 60 amino acids which are homologous to the DED of FADD (FIGS. 1B, 2A). A BLAST search revealed that residues 7-75 and 101-169 of FLICE matched the DED of FADD (residues 4-76) and shared 39% identity (55% similarity) and 28% identity (55% similarity), respectively. Another protein, designated PEA-15, was also identified as a DED-containing protein by BLAST search. PEA-15 is an astrocytic phosphoprotein of unknown function (H. Cheneiweiss, unpublished; accession number S55384).
FLICE is a Novel Member of the ICE/CED-3 Family
While the N-terminus of FLICE contains the FADD homology domains, the remainder of the protein is highly homologous to the ICE/CED-3 family, particularly in the regions corresponding to the active subunits of ICE (FIGS. 2B-1 and 2B-2). Phylogenetic analysis of the ICE/CED-3 gene family showed that FLICE is a member of the CED-3 subfamily which includes Yama/CPP32, Mch2, ICE-LAP3, and ICE-LAP6 (FIG. 2C). Like C. elegans CED-3, FLICE contains a long N-terminal putative pro-domain, but in this case, importantly, the pro-domain shares homology to the DED of FADD. It is likely that CAP3 represents this pro-domain since its estimated MW (26 kDa) by 2D gel analysis corresponds exactly with the calculated MW of the putative FLICE pro-domain (aa1-220). There was no DED homology present in any of the other ICE/CED-3 family members.
Based on the x-ray crystal structure of ICE. (Walker et al., 1994; Wilson et al., 1994), the amino acid residues His.sup.237, Gly.sup.238, Cys.sup.285 of ICE are involved in catalysis, while the residues Arg.sup.179, Gln.sup.283 and Arg.sup.341 form a binding pocket for the carboxylate side chain of the P.sub.1 aspartic acid. These six residues are conserved in all ICE/CED-3 family members thus far cloned as well as in FLICE. However, residues that form the P.sub.2 -P.sub.4 binding pockets are not widely conserved among family members, suggesting that they may determine substrate specificity. Interestingly, FLICE contains a unique pentapeptide QACQG instead of the QACRG shared by most other family members (FIGS. 2B-1 and 2B-2) with the exception of the recently cloned ICE-LAP6, which contains a QACGG pentapeptide (Duan et al, 1996b).
Tissue Distribution of FLICE
Northern blot analysis revealed that FLICE is constitutively expressed in many fetal and adult human tissues, but not in the fetal brain. Interestingly, there was relatively higher expression of FLICE in peripheral blood leukocytes which is consistent with the important role of CD95 signaling in lymphocyte homeostasis (Nagata and Golstein, 1995). The mRNA transcript was approximately 3.0 kb.
FLICE Associates with the DED of FADD
Previous studies showed that FADD-DN (missing the DED) blocked the recruitment of CAP4 to the DISC, suggesting that the DED was an essential component. (Chinnaiyan et al., 1996a). To determine whether FLICE can directly bind FADD and whether the DED of FADD is necessary for this binding, in vitro transcribed/translated FLICE was precipitated with His.sub.6 -tagged FADD and His.sub.6 -tagged FADD-DN immobilized onto Ni.sup.2+ beads. As predicted, FLICE bound full-length FADD but not FADD-DN. Further confirmation was obtained by using 293T cell-generated proteins. In this case, 293T cells were transiently transfected with C-terminal tagged FADD-AU1 and .DELTA.FADD-AU1, in which the 18 N-terminal amino acids of the DED are deleted. Cell lysates were then immunoprecipitated with anti-AU1 antibody and protein G-coupled sepharose. The beads were subsequently incubated with .sup.35 S-labeled FLICE or Yama/CPP32, a related ICE/CED-3 family member. Consistent with the results, FLICE, but not Yama, bound full-length FADD.
Granzyme .beta.-activated FLICE Cleaves PARP
Members of the ICE/CED-3 gene family are synthesized as pro-enzymes and activated by proteolytic cleavage at specific aspartate residues to form heterodimeric enzymes. In ICE, this cleavage removes the pro-domain and produces a heterodimeric complex consisting of p20 and p10 subunits. (Thornberry et al., 1992). Similarly, activated Yama is comprised of two subunits, p17 and p12, which are derived from a 32 kDa pro-enzyme. (Nicholson, 1996). Recent studies on granzyme B suggest that cytotoxic lymphocytes may utilize this secreted serine protease to directly activate members of the ICE/CED-3 family. It has previously been demonstrated that granzyme B can proteolytically activate pro-Yama, generating an active enzyme capable of cleaving the death substrate PARP into characteristic fragments. (Darmon et al., 1995; Quan et al., 1996). By contrast, ICE, although cleaved by granzyme B, fails to be activated. (Quan et al., 1996).
Thus, we determined whether FLICE can serve as a substrate for granzyme B, and more importantly, whether FLICE can function as a cysteine protease. In vitro transcribed/translated FLICE and Yama were incubated with purified granzyme B. (Hanna et al., 1993). After 4 hrs at 37.degree. C., FLICE and Yama were proteolytically processed, generating a putative active p20/p10 and p17/p12 subunits, respectively. Next, we assessed whether granzyme B-mediated cleavage of FLICE generates an active enzyme by assaying for PARP cleavage. PARP is proteolyzed during many forms of apoptosis and the enzyme(s) responsible likely belong to the ICE/CED-3 family. (Nicholson et al., 1995; Tewari et al., 1995). To exclude the possibility of direct cleavage of PARP by granzyme B, granzyme B-processed FLICE and Yama were incubated with a selective inhibitor of granzyme B (anti-GraB) as previously described (Quan et al., 1996). Importantly, both granzyme B-processed Yama and FLICE were active as determined by their ability to cleave PARP. Therefore, unlike ICE, FLICE and other members of the CED-3 subfamily are able to cleave PARP into signature apoptotic fragments. (Tewari et al., 1995).
Overexpression of FLICE Induces Apoptosis which Is Abrogated by ICE Family Inhibitors
To study the functional role of FLICE, we transiently transfected MCF7 breast carcinoma cells with an expression vector encoding the FLICE protein and subsequently assessed for apoptotic features. We chose MCF7 cells since they are easily transfectable and are sensitive to CD95-induced killing. (Tewari and Dixit, 1995b). Like the other ICE/CED-3 family members, overexpresion of FLICE causes apoptosis (FIG. 3). The FLICE-transfected cells displayed morphological alterations typical of adherent cells undergoing apoptosis, becoming rounded, condensed, and detaching from the dish. FLICE-induced cell death was inhibited by the broad spectrum ICE inhibitor z-VAD-fmk. (Fearnhead et al., 1995; Pronk et al., 1996). (FIG. 3), which is also a potent inhibitor of CD95-induced apoptosis. Like the peptide ICE family inhibitor, CrmA blocked FLICE-induced cell death (FIG. 3), suggesting that FLICE may be a physiologic target for this poxvirus serpin. Alternatively, FLICE may activate a downstream CrmA-sensitive protease.
Discussion
The Physical Link Between The Death Receptors and the Apoptotic Proteases
Prior to this study, the link between activators and effectors of the cell death machinery was unclear. Here we demonstrate that a cell surface death receptor (CD95) uses an adaptor molecule (FADD) to physically engage a cytosolic apoptotic protease termed FLICE. The death domain is an important signaling motif shared by both CD95 and TNFR-1 and oligomerization of this domain recruits cytosolic adaptor proteins to assemble a signalling complex (DISC) (Peter et al., 1996). The assembly of a DISC is essential for CD95 signal transduction. Upon activation, the death domain of the receptor binds to the death domain of the adaptor molecule FADD (CAP1,2) and thereby recruits it to form part of the DISC. The complete DISC is created when FADD, in turn, binds and recruits the ICE/CED-3-like protease FLICE (CAP4) or ICE LAP-7.
While the main activity of CD95 is to trigger cell death, TNFR-1 can signal a diverse range of activities including fibroblast proliferation, resistance to intracellular pathogens including chlamidiae, and synthesis of prostaglandin E2 (Tartaglia and Goeddel, 1992). Likewise, TNFR-1 recruits a multivalent adaptor molecule termed TRADD, which, like FADD, contains a death domain required for receptor association (Hsu et al., 1995). TRADD has been shown to bind a number of signaling molecules including FADD, TRAF2 and RIP. (Hsu et al., 1996a; Hsu et al., 1996b). A dominant negative version of FADD blocks TNF killing. (Chinnaiyan et al., 1996a; Hsu et al., 1996a), while dominant negative versions of TRAF2 and RIP block TNF-induced NF-KB activation. (Hsu et al., 1996a; Hsu et al., 1996b). This indicates the existence of a signaling bifurcation dictated by the nature of the associated adaptor molecules. Regardless, TNFR-1 indirectly uses FADD to engage the death protease FLICE and thereby unites the death pathways that emanate from CD95 and TNFR-1.
Emphasizing the evolutionary conservation of the apoptotic machinery, the Drosophila death protein Reaper shares weak homology to the death domain motif. (Golstein et al., 1995) and, like CD95 and TNFR-1, is capable of initiating the apoptotic program. (White et al., 1994; White et al., 1996). Reaper mediates its actions by activating ICE/CED-3-like proteases as both z-VAD-fmk and the baculovirus ICE-family inhibitor p35 block Reaper-induced cell death. (Pronk et al., 1996; White et al., 1996). Given our data on death domain receptors, it is tempting to speculate that Reaper may exert its actions by directly or indirectly engaging ICE/CED-3-like proteases in a manner analogous to FADD and CD95, respectively.
FLICE is Homologous to FADD and is a Cysteine Protease of the ICE/CED-3 Family
FLICE is a molecule with homology to both FADD and the ICE/CED-3 family. The pro-domain of FLICE is homologous to the DED of FADD, which is a critical motif required for engaging the death pathway. Phylogenetic comparison of FLICE with other ICE/CED-3 family members classify this cysteine protease in the CED-3 subfamily along with Yama/CPP32, ICE-LAP3, ICE-LAP6, and Mch2 (FIG. 2C). FLICE clearly represents the most upstream enzymatic activity in the CD95 pathway and it will thus be important to determine which ICE/CED-3 family members are proteolytically activated by FLICE, leading to amplification of the death signal. Though activated FLICE cleaves PARP well, this is unlikely to be the physiologic substrate for FLICE and was only used as a readout to monitor the generation of proteolytically competent FLICE by granzyme B. Physiologic PARP cleavage is more likely mediated by the downstream enzymes Yama and ICE-LAP3. (Fernandes-Alnemri et al., 1995; Lippke et al., 1996; Nicholson et al., 1995; Tewari et al., 1995).
Importantly, the Asp specific pro-apoptotic serine protease granzyme B proteolytically activates FLICE in a manner similar to Yama/CPP32. (Darmon et al., 1995; Quan et al., 1996). By contrast, ICE is processed, but not activated by granzyme B (Quan et al., 1996). Secretion of granzyme B, in combination with the pore-forming protein perforin, is one of two mechanisms by which cytotoxic T lymphocytes (CTLs) trigger apoptosis of susceptible target cells, the other being engagement of CD95 on the target cell surface. (Berke, 1995). Thus, granzyme B likely mediates apoptosis by directly activating the target cell's death effector machinery, which is composed of an arsenal of intracellular, CED-3-like cysteine proteases. FLICE and other members of the CED-3 subfamily (K. Orth, A. M. C., unpublished observation) are almost certainly activated during both granzyme B- and CD95-mediated apoptosis, suggesting the convergence of distinct apoptotic pathways involved in cell-mediated immunity.
Identity of CAP3
CAP3 likely represents the N-terminal pro-domain of FLICE consistent with the finding that the predicted molecular weight and pI of the N-terminal 220 amino acids of FLICE correspond to CAP3. Like CAP4, CAP3 was only found associated with the activated CD95 receptor requiring full-length FADD for binding (Chinnaiyan et al., 1996a). It is conceivable that CAP3 is generated by proteolytic activation of FLICE during CD95 ligation, but retained in the DISC by virtue of its ability to still bind the DED of FADD. The active dimeric p20/p10 subunits of FLICE are likely liberated from the DISC and are free to cleave and activate downstream apoptotic proteases. If this scenario is correct, CAP4 represents the inactive zymogen form of FLICE, which during CD95-signaling gets activated, generating a free, active heterodimeric enzyme and a pro-domain that is still retained by the DISC (CAP3).
How is FLICE activated?
Taken together, our data enables us to propose a model for FLICE activation. In the latent state, the two DEDs of FLICE may bind to each other preventing FLICE activation. CD95 stimulation and resultant trimerization causes binding of FADD. This may trigger a conformational change in the DED of FADD allowing it to bind one of the corresponding DEDs of FLICE. By disrupting the self-association of the FLICE DEDs, the ICE/CED-3 homology domain of FLICE may be free to undergo autocatalytic activation. The active p20/p10 protease is liberated from the DISC and subsequently ignites a proteolytic cascade composed of other ICE/CED-3 family members. The FLICE pro-domain (CAP3) remains bound to the receptor.
Conclusions
The CD95-mediated apoptotic cascade is initiated by the direct physical association of the death receptor CD95 with the adaptor molecule FADD and the effector protease FLICE, a novel member of the ICE/CED-3 family. The assembly of this signaling complex occurs in a hierarchical manner upon receptor activation. The death domain of the receptor binds to the death domain of the adaptor molecule FADD, which, in a manner that remains to be determined, binds and activates FLICE to generate the most apical enzymatic component of a death cascade composed of other ICE/CED-3 family members. For the first time, the essential components of an apoptotic cascade have been elucidated.
Clearly, there is a need for factors that are useful for inducing apoptosis for therapeutic purposes, for example, as an antiviral agent, an anti-tumor agent and to control embryonic development and tissue homeostasis, and the roles of such factors in dysfunction and disease. There is a need, therefore, for identification and characterization of such factors that are interleukin-1 beta converting enzyme apoptosis proteases, and which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
The polypeptide of the present invention has conserved residues of interleukin-1 beta converting enzyme apoptosis proteases, and have amino acid sequence homology to known interleukin-1 beta converting enzyme apoptosis proteases.